Apoptotic protease Mch6, nucleic acids encoding same and methods of use

ABSTRACT

The invention provides an isolated gene encoding Mch6 as well as functional fragments thereof. Also provided are isolated nucleic acid sequences encoding Mch6 or functional fragments thereof. The gene or nucleic acid sequences can be single or double stranded nucleic acids corresponding to coding or non-coding strands of the Mch6 nucleotide sequences. The invention further provides an isolated Mch6 polypeptide and isolated large and small subunits of the Mch6 polypeptide, including functional fragments thereof.

[0001] This invention was made with government support under researchgrant AI 35035 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0002] The present invention relates generally to apoptosis, orprogrammed cell death, and more particularly, to a novelaspartate-specific cysteine protease that can be used to modulateapoptosis for the therapeutic treatment of human diseases.

[0003] Apoptosis is a normal physiological process of cell death thatplays a critical role in regulating tissue homeostasis by ensuring thatthe rate of new cell accumulation produced by cell division is offset bya commensurate rate of cell loss due to cell death. It has now becomeclear that disturbances in apoptosis, also referred to as physiologicalcell death or programmed cell death, which prevent or delay normal cellturnover, can be just as important to the pathogenesis of diseases asknown abnormalities in the regulation of proliferation and the cellcycle. Like cell division, which is controlled through complexinteractions between cell cycle regulatory proteins, apoptosis issimilarly regulated under normal circumstances by the interaction ofgene products that either induce or inhibit cell death.

[0004] The stimuli that regulate the function of these apoptotic geneproducts include both extracellular and intracellular signals. Eitherthe presence or the removal of a particular stimulus can be sufficientto evoke a positive or negative apoptotic signal. For example,physiological stimuli that prevent or inhibit apoptosis include, forexample, growth factors, extracellular matrix, CD40 ligand, viral geneproducts, neutral amino acids, zinc, estrogen and androgens. Incontrast, stimuli that promote apoptosis include growth factors such astumor necrosis factor (TNF), Fas, and transforming growth factor β(TGFβ). Other stimuli that promote apoptosis include, for example,neurotransmitters, growth factor withdrawal, loss of extracellularmatrix attachment, intracellular calcium and glucocorticoids. Otherstimuli, including those of environmental and pathogenetic origins, caneither induce or inhibit programmed cell death. Although apoptosis ismediated by diverse signals and complex interactions of cellular geneproducts, the results of these interactions ultimately feed into a celldeath pathway that is evolutionarily conserved between humans andinvertebrates.

[0005] Several gene products that modulate the apoptotic process havenow been identified. Although these products can be generally separatedinto two basic categories, gene products from each category can functionto either inhibit or induce programmed cell death. One family of geneproducts is related to the protein Bcl-2, which inhibits apoptosis whenoverexpressed in cells. Other members of this gene family include, forexample, Bax, Bak, Bcl-x_(L), Bcl-x_(S), and Bad. While some of theseproteins can prevent apoptosis, others augment apoptosis, for example,Bcl-x_(S) and Bak, respectively.

[0006] A second family of gene products, the aspartate-specific cysteineproteases (ASCPs), are genetically related to the ced-3 gene product,which was initially shown to be required for programmed cell death inthe roundworm, C. elegans. The ASCP family of proteases includes humanICE (interleukin-1-β converting enzyme), ICH-1_(L), ICH-1_(S), CPP32,Mch2, Mch3, Mch4, Mch5, ICH-2 and ICE_(rel)-III Among the commonfeatures of these gene products are that 1) they are cysteine proteaseswith specificity for substrate cleavage at Asp-x bonds, 2) they share arelatively conserved pentapeptide sequence, QACRG (SEQ ID NO:79) orQACQG (SEQ ID NO:80), within the active site and 3) they are synthesizedas proenzymes that require proteolytic cleavage at specific aspartateresidues for activation of protease activity. In the case of ICE,cleavage of the proenzyme produces two polypeptide protease subunits ofapproximately 20 kDa, known as p20, and 10 kDa, known as p10, thatcombine non-covalently to form a tetramer comprising two p20:p10heterodimers. Although these proteases induce cell death when expressedin cells, several alternative structural forms, such as ICEδ, ICEε,ICH-1_(S) and Mch2β, actually function to inhibit apoptosis.

[0007] In addition to the Bcl-2 and ASCP gene families that play a rolein apoptosis in mammalian cells, it has become increasingly apparentthat other gene products that are important in mammalian cell death haveyet to be identified. For example, in addition to Ced-3, another C.elegans gene known as Ced-4 is also required for programmed cell deathin C. elegans. However, mammalian homologs of Ced-4 remain elusive andhave not yet been identified. Further, it is ambiguous whether othergenes belong to either of the above two apoptotic gene families or whatrole they may play in the programmed cell death pathway. Finally, it isunclear what physiological control mechanisms regulate programmed celldeath or how the cell death pathways interact with other physiologicalprocesses within the organism. For example, it has recently beensuggested that cytotoxic T-lymphocytes mediate their destructivefunction by inducing apoptosis in their target cells.

[0008] Apoptosis maintains tissue homeostasis in a range ofphysiological processes such as embryonic development, immune cellregulation and normal cellular turnover. Therefore, the dysfunction orloss of regulated apoptosis can lead to a variety of pathologicaldisease states. For example, the loss of apoptosis can lead to thepathological accumulation of self-reactive lymphocytes such as thatoccurring with many autoimmune diseases. Inappropriate loss of apoptosiscan also lead to the accumulation of virally infected cells and ofhyperproliferative cells such as neoplastic or tumor cells. Similarly,the inappropriate activation of apoptosis can also contribute to avariety of pathological disease states including, for example, acquiredimmunodeficiency syndrome (AIDS), neurodegenerative diseases andischemic injury. Treatments that are specifically designed to modulatethe apoptotic pathways in these and other pathological conditions canchange the natural progression of many of these diseases.

[0009] Thus, there exists a need to identify new apoptotic genes andtheir gene products to modulate apoptosis for the therapeutic treatmentof human diseases. The present invention satisfies this need andprovides related advantages as well.

SUMMARY OF THE INVENTION

[0010] The invention provides an isolated gene encoding Mch6 as well asfunctional fragments thereof. Also provided are isolated nucleic acidsequences encoding Mch6 or functional fragments thereof. The gene ornucleic acid sequences can be single or double stranded nucleic acidscorresponding to coding or non-coding strands of the Mch6 nucleotidesequences. The invention further provides an isolated Mch6 polypeptideand isolated large and small subunits of the Mch6 polypeptide, includingfunctional fragments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIGS. 1a, 1 b and 1 c show the nucleotide and predicted amino acidsequence of Mch6, listed as SEQ ID NO:1 and SEQ ID NO:2, respectively.The active site pentapeptide sequence QACGG (SEQ ID NO:78) isunderlined. Cleavage sites after Asp315 and Asp330 are indicated byvertical arrows.

[0012]FIG. 2 shows a multiple amino acid sequence alignment ofrelatively conserved regions within the ASCPs. The ASCPs are Mch6 (SEQID NO:6, consisting of noncontiguous SEQ ID NOs:17-22), Mch5 (SEQ IDNO:7, consisting of noncontiguous SEQ ID NOs:23-27), Mch4 (SEQ ID NO:8,consisting of noncontiguous SEQ ID NOs:28-32), Mch3 (SEQ ID NO:9,consisting of noncontiguous SEQ ID NOs:33-37), Mch2 (SEQ ID NO:10,consisting of noncontiguous SEQ ID NOs:38-43), CPP32 (SEQ ID NO:11,consisting of noncontiguous SEQ ID NOs:44-48), CED-3 (SEQ ID NO:12,consisting of noncontiguous SEQ ID NOs:49-53), ICE (SEQ ID NO:13,consisting of noncontiguous SEQ ID NOs:54-59), TX (SEQ ID NO:14,consisting of noncontiguous SEQ ID NOs:60-65), ICErelIII (SEQ ID NO:15,consisting of noncontiguous SEQ ID NOs:66-71) and ICH-1 (SEQ ID NO:16,consisting of noncontiguous SEQ ID NOs:72-77).

[0013] Based on the crystal structure of ICE, specific residues areindicated by lowercase letters below the sequences: “c” for residuesinvolved in catalysis, “b” for residues that bind thesubstrate-carboxylate of P1 Asp and “a” for residues adjacent to thesubstrate P2-P4 amino acids. “DX” indicates known and potentialprocessing sites between the small and large subunits of ASCPs. Theroman numerals on the left indicate the three ASCP-subfamilies: theCed-like subfamily (I), the ICE-like subfamily (II) and the Nedd2/Ich-1subfamily (III). The asterisk indicates the nonconservative substitutionin the active site pentapeptide sequences of Mch4, Mch5 and Mch6.

[0014]FIG. 3 shows a schematic diagram illustrating the processing ofproMch6. proMch6 can be processed by CPP32 after Asp330. proMch6 canalso be processed preferentially after Asp315 by granzyme B to generatethe large subunit, known as p35, and the small subunit, known as p10, ofmature Mch6. The active site pentapeptide QACGG (SEQ ID NO:78) in thelarge subunit is indicated.

DETAILED DESCRIPTION OF THE INVENTION

[0015] This invention is directed to a novel apoptotic protease termedMch6 (mammalian ced-3 homolog 6). Mch6 is a member of theaspartate-specific cysteine protease (ASCP) family of proteases thatincludes, for example, ICE (Alnemri et al., J. Biol. Chem. 270:4312-4317(1995)), CPP32 (Fernandes-Alnemri et al., J. Biol. Chem. 269:30761-30764(1994)), Nedd2/Ich-1 (Kumar et al., Genes & Development 8:1613-1626(1994); Wang et al., Cell 78:739-750 (1994)), Mch2 (Fernandes-Alnemri etal., Cancer Res. 55:2737-2742 (1995)), Mch3 (Fernandes-Alnemri et al.,Cancer Res. 55:6045-6052 (1995), Mch4 (Fernandes-Alnemri et al., Proc.Natl. Acad. Sci. USA 93:7464-7470 (1996)), Mch5 (Fernandes-Alnemri etal. (1996) supra), TX (ICH-2, ICErel-II) (Faucheu et al., EMBO14:1914-1922 (1995); Kamens et al., J. Biol. Chem. 270:15250-15256(1995); Munday et al., J. Biol. Chem. 270:15870-15876 (1995)) andICErel-III (Munday et al. (1995) supra).

[0016] Mch6 shares amino acid sequence homology with several ASCPs, butits catalytic site QACGG (SEQ ID NO:78) differs in the fourth residuefrom the relatively conserved catalytic sites in other known ACSPs(FIGS. 1 and 2).

[0017] Like many ASCPs, Mch6 is synthesized as a proenzyme, which can beproteolytically cleaved by, for example, CPP32 or granzyme B. Thecleavage of Mch6 by these two enzymes is described further below inExamples III and IV, respectively. Cleavage of Mch6 yields two subunits,a large subunit of approximately 35 kDa and a small subunit ofapproximately 10 kDa, which associate to form an active heterodimercomplex. Like other ASCPs, the active Mch6 complex can act as a proteaseand requires an Asp residue in the P1 position of the substrate bindingsite with a small, preferably hydrophobic, residue in the P1′ position.

[0018] In one embodiment, the invention is directed to nucleic acidsencoding the apoptotic protease Mch6. The nucleic acids are used toproduce the recombinant Mch6 ASCP protease. The recombinant polypeptidescan be used to screen for Mch6 inhibitors. Mch6 inhibitors include thosethat inhibit protease activity as well as compounds that inhibit Mch6binding to other polypeptides. Such compounds are useful aspharmaceuticals for treating or preventing diseases characterized byapoptotic cell death. Alternatively, the Mch6 polypeptides can be usedto screen for compounds that activate or act as agonists of Mch6, suchas by inducing cleavage of the proenzyme into its active subunits. Suchcompounds are similarly useful as pharmaceuticals for treating orpreventing diseases characterized by the loss of apoptotic cell death.

[0019] As used herein, the term “substantially” when referring to a Mch6nucleotide or amino acid sequence is intended to refer to the degree towhich two sequences of between about 15-30 or more nucleotides in lengthare identical or similar, so as to be considered by those skilled in theart to be functionally equivalent. For example, the Mch6 nucleic acid ofthe invention has a nucleotide sequence substantially the same as thatshown in FIG. 1 and as SEQ ID NO:1. Thus, if a second sequence isconsidered by those skilled in the art to be functionally equivalent tothe sequence shown as SEQ ID NO:1, then the second sequence issubstantially the same as that shown as SEQ ID NO:1. Methods forsequence comparisons and determinations of similarity are well known androutine within the art.

[0020] Functionally equivalent nucleic acid sequences include, forexample, sequences that are related, but different and encode the sameMch6 polypeptide due to the degeneracy of the genetic code as well assequences that are related, but different and encode a different Mch6polypeptide that exhibits similar functional activity. In both cases,the nucleic acids encode functionally equivalent gene products.Functional fragments of Mch6 encoding nucleic acids such asoligonucleotides, polynucleotides, primers and the like are alsoconsidered to be within the definition of the term and the invention asclaimed. Functional equivalency is also relevant to Mch6 nucleic acidsthat do not encode gene products, for example, but instead arefunctional elements in and of themselves. Specific examples of suchfunctional nucleic acids include, for example, promoters, enhancers andother gene expression and transcription regulatory elements.

[0021] An Mch6 polypeptide of the invention has an amino acid sequencesubstantially similar to that shown in FIG. 1 and in SEQ ID NO:2.Functionally equivalent Mch6 amino acid sequences similarly includes,for example, related, but different sequences so long as the differentpolypeptide exhibits at least one functional activity of Mch6. Suchrelated, but different polypeptides include, for example, substitutionsof conserved and nonessential amino acids. Fragments and functionaldomains of Mch6 are similarly included within the definition of the termand the claimed invention.

[0022] Therefore, it is understood that limited modifications may bemade without destroying the biological function of the Mch6 polypeptideand that only a portion of the entire primary structure may be requiredin order to effect activity. For example, minor modifications of theMch6 amino acid sequence (SEQ ID NO:2) that do not destroy theiractivity also fall within the definition of Mch6 and within thedefinition of the polypeptide claimed as such. Also within thedefinition of the claimed polypeptides are, for example, geneticallyengineered fragments of Mch6, either alone or fused to heterologousproteins such as fusion proteins that retain measurable enzymatic orother biological activity.

[0023] It is understood that minor modifications of primary amino acidsequence may result in polypeptides that have substantially equivalentor enhanced function as compared to the sequences set forth in FIG. 1(SEQ ID NO:2). These modifications may be deliberate, as throughsite-directed mutagenesis, or may be accidental such as through mutationin hosts that are Mch6 producers. All of these modifications areincluded as long as Mch6 biological function is retained. Further,various molecules can be attached to Mch6, for example, other proteins,carbohydrates, lipids, or chemical moieties. Such modifications areincluded within the definition of an Mch6 polypeptide.

[0024] The invention provides a gene encoding Mch6, or fragment thereof.The invention also provides an isolated nucleic acid sequence encodingMch6, or fragment thereof. The gene and nucleic acid sequences encodesubstantially the sequence as shown in SEQ ID NO:1. Fragments of thegene or nucleic acid sequence are provided that comprise single ordouble stranded nucleic acids having substantially the sequences shownin SEQ ID NO:1.

[0025] The Mch6 nucleic acid of the present invention was identified andisolated by a novel approach of searching a human database of expressedsequence tags (ESTs) under various stringencies to identify potentiallynew sequence fragments that may have homology to the ICE family ofcysteine proteases. Previously these proteases were referred to as theICE-family of proteases and thus the initial search criteria wasdirected to the ICE family of cell death proteases. However, with therecent identification of Mch4 and Mch5, the proteases have beenreclassified into three subfamilies referred to herein as the Ced-like,ICE-like and Nedd2/ICH-1-like subfamilies of cell death proteases.

[0026] When searching for potential new sequences related to the ICEfamily of proteases, novel sequences are identified by their homology tothe ICE family of cell death proteases. These novel sequences are thenused to design primers for attempting PCR amplification and cloning ofthe actual cDNA. The second primer for the amplification is designed toencompass homologous regions in nucleic acid sequences that encode knownICE protease family members. In this specific case, the primer wasdirected to a sequence analogous to the GSWFI/GSWYI pentapeptidesequence that is conserved in a number of the ICE/Ced-3 family ofproteases. The primer design should take into account the predictedstrandedness of both the EST sequence primer and the known primer. Thus,only if the homology search and primer hybridization conditions aresuccessfully determined will such an approach allow PCR amplification ofa fragment of the putative novel protease cDNA.

[0027] Because searching a genetic data base will yield homologoussequence matches to any nucleotide sequence query, additional criteriamust be used to identify the authentic ICE subfamily homolog from amongthe non-specific homology matches. ICE family members share the highestdegree of homology in the active site and catalytically important aminoacid residues. A given EST returned by the search may not include one ofthese highly homologous sites, but may only include a region within theprotease with cryptic homology. Confirming an EST as a novel ICEprotease involves translation of all the positive EST hits in threedifferent reading frames and subsequent identification of conservativeactive site or catalytically important amino acid sequence motifs. Then,using conventional cDNA cloning, a full length cDNA of the putativenovel protease can be obtained and 1) analyzed for overall structuralhomology to ICE family members, 2) recombinantly expressed and analyzedfor cysteine protease activity, and 3) analyzed for the induction ofprogrammed cell death by heterologous expression of the cDNA inappropriate cells.

[0028] In addition to the methods described above for isolating Mch6encoding nucleic acids, alternative methods can similarly be employed.For example, using the teachings described herein, those skilled in theart can routinely isolate and manipulate Mch6 nucleic acids usingmethods well known in the art. All that is necessary is the sequence ofthe Mch6-encoding nucleic acid (FIG. 1 and SEQ ID NO:1) or its aminoacid sequence (FIG. 1 and SEQ ID NO:2). Such methods include, forexample, screening a cDNA or genomic library by using syntheticoligonucleotides, nucleic acid fragments or primers as hybridizationprobes. Alternatively, antibodies to the Mch6 amino acid sequence orfragments thereof can be generated and used to screen an expressionlibrary to isolate Mch6-encoding nucleic acids. Other binding reagentsto an Mch6 polypeptide can similarly be used to isolate an Mch6polypeptide having substantially the amino acid sequence shown inFIG. 1. Similarly, substrate reagents such as non-cleavable peptideanalogs of cysteine proteases can be used to screen and isolate an Mch6polypeptide.

[0029] In addition, recombinant DNA methods currently used by thoseskilled in the art include the polymerase chain reaction (PCR). Whencombined with the Mch6 nucleotide and amino acid sequences describedherein, PCR allows reproduction of Mch6 encoding sequences. PCR canamplify desired sequences exponentially starting from as little as asingle gene copy. The PCR technology is the subject matter of U.S. Pat.Nos. 4,683,195, 4,800,159, 4,754,065 and 4,683,202, all of which areincorporated by reference herein.

[0030] The above-described methods are known to those skilled in the artand are described, for example, in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, NY (1992) and thevarious references cited therein and in Ansubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.(1989); and in Harlow et al., Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, NY (1989). These references and thepublications cited therein are hereby expressly incorporated herein byreference.

[0031] The invention provides an isolated Mch6 polypeptide comprisingsubstantially the amino acid sequence as that shown in FIG. 1 (SEQ IDNO:2). Mch6 functional fragments are also provided. Specific examples ofMch6 functional fragment include, for example, the catalytic domain thatcontains the active site amino acid sequence QACGG (SEQ ID NO:78). Whencompared to the active site amino acid sequence of other ASCP familymembers, QACRG (SEQ ID NO:79) or QACQG (SEQ ID NO:80), this active sitesequence is similar but differs at position 4 with Arg (R) substitutedby Gly (G).

[0032] An isolated Mch6 polypeptide of the invention can be obtained bya variety of methods known within the art. For example, the isolatedpeptides can be purified by biochemical methods including affinitychromatography, for example. Affinity matrices for Mch6 isolation can beanti-Mch6 monoclonal or polyclonal antibodies prepared against the aminoacid sequence shown in FIG. 1 (SEQ ID NO:2) or against fragments thereofsuch as synthetic peptides. Alternatively, substrate analogues orenzymatic inhibitors of Mch6 can similarly be used as affinity matricesto isolate substantially pure a Mch6 polypeptide of the invention.

[0033] An Mch6 polypeptide can also be produced by recombinant methodsknown to those skilled in the art. Recombinant Mch6 polypeptidesinclude, for example, an amino acid sequence substantially the same asthat shown in FIG. 1 (SEQ ID NO:2), as well as fusion proteins andfragments thereof. The Mch6-encoding nucleic acids can be cloned intothe appropriate vectors for propagation, manipulation and expression.Such vectors are known or can be constructed by those skilled in the artand should contain all expression elements necessary for thetranscription, translation, regulation, and if desired, sorting of theMch6 polypeptides. The vectors can also be for use in either procaryoticor eucaryotic host systems so long as the expression and regulatoryelements are compatible. One of ordinary skill in the art will knowwhich host systems are compatible with a particular vector. Therecombinant polypeptides produced can be isolated by the methodsdescribed above.

[0034] The invention further provides isolated large and small subunitsof an Mch6 polypeptide. For example, the proMch6 polypeptide can beproteolytically cleaved by CPP32 or granzyme B to form a large and smallsubunit (Examples III and IV). In particular, CPP32 can cleave proMch6into a large subunit having an approximate molecular weight of 37 kDa(p37) and a small subunit having an approximate molecular weight of 10kDa (p10). Similarly, granzyme B can cleave proMch6 into a large subunithaving an approximate molecular weight of 35 kDa (p35) and a smallsubunit having an approximate molecular weight of 12 kDa (p12).Moreover, other components of the apoptotic pathway can process Mch6into a larger and a smaller cleavage product. Accordingly, the terms“large subunit” and “small subunit” will readily be understood to referto any larger proteolytic cleavage product such as p37 or p35, and anysmaller cleavage product such as p10 or 12, respectively.

[0035] Apoptosis plays a significant role in numerous pathologicalconditions in that programed cell death is either inhibited, resultingin increased cell survival, or enhanced, which results in the loss ofcell viability. Examples of pathological conditions resulting fromincreased cell survival include cancers such as lymphomas, carcinomasand hormone-dependent tumors. Such hormone-dependent tumors include, forexample, breast, prostate and ovarian cancer. Increased cell survival orapoptosis inhibition can also result in autoimmune diseases such assystemic lupus erythematosus and immune-mediated glomerulonephritis, aswell as viral infections such as herpesvirus, poxvirus and adenovirus.

[0036] In contrast, apoptotic diseases where enhanced programed celldeath is a prevalent cause generally includes, for example, degenerativedisorders such as Alzheimer's disease, Parkinson's disease, amyotrophiclateral sclerosis, retinitis pigmentosa, and cerebellar degeneration.Other diseases associated with increased apoptosis include, for example,myelodysplastic syndromes such as aplastic anemia and ischemic injury,including myocardial infarction, stroke and reperfusion injury.

[0037] The Mch6-encoding nucleic acids and polypeptides of the inventioncan be used to diagnose, treat or reduce the severity of celldeath-mediated diseases such as those described above as well as otherdiseases mediated by either increased or decreased programmed celldeath. Additionally, the Mch6-encoding nucleic acids and polypeptides ofthe invention can be used to screen for pharmaceutical compounds andmacromolecules that inhibit or promote Mch6-mediated apoptosis.

[0038] For example, the Mch6-encoding nucleic acids, polypeptides andfunctional fragments thereof can be used to diagnose or generatereagents to diagnose diseases mediated or characterized by programedcell death. Diagnosis can be by nucleic acid probe hybridization withMch6-containing nucleotide sequences, antibody- or ligand-mediateddetection with Mch6-binding agents, or by enzyme catalysis of detectableMch6 substrates. Such methods are routine to those skilled in the art.Detection can be performed ex vivo, for example, by removing a cell ortissue sample from an individual exhibiting or suspected of exhibiting acell death-mediated disease. Correlation of increased Mch6 expression oractivity is indicative of diseases characterized by enhanced programmedcell death, whereas correlation of decreased Mch6 expression or activityis indicative of diseases characterized by the inhibition of programmedcell death.

[0039] The above Mch6 polypeptide can also be formulated intopharmaceutical compositions for treating cell death-mediated diseasescharacterized by increased cell survival and proliferation. Functionalfragments and peptides such as the catalytic domain of Mch6 cansimilarly be formulated to treat such diseases. Additionally, moleculesthat interact with Mch6 can also be used to induce Mch6-mediatedapoptosis. Administration of Mch6 polypeptides and functional fragmentsthereof will induce apoptosis in treated cells and eliminate those cellscharacterized by increased cell survival or proliferation. Non-Mch6polypeptides that do not directly act on Mch6 substrates but induce theactivation of the Mch6 protease can similarly be administered to treatdiseases characterized by increased cell survival and proliferation.

[0040] To be effective, the Mch6 polypeptide must be introduced into thecells by means characterized by increased cell survival. Introductioncan be accomplished by a variety of means known within the artincluding, for example, lipid vesicles and receptor-mediatedendocytosis. Targeting to the appropriate cell type can similarly beaccomplished through conjugation to specific receptor ligands, specifictarget cell antibodies and the like.

[0041] The Mch6 polypeptide is administered by conventional methods indosages that are sufficient to induce apoptosis in the cellscharacterized by increased cell survival or proliferation. Such dosagesare known or can be easily determined by those skilled in the art.Administration can be accomplished, for example, by intravenous,interperitoneal or subcutaneous injection. Administration can beperformed in a variety of different regimes that include single highdose administration, repeated small dose administration, or acombination of both. The administration will depend on the cell type,progression of the disease and overall health of the individual and willbe known or can be determined by those skilled in the art.

[0042] In contrast to the induction of Mch6-mediated apoptosis to treatpathological conditions characterized by increased cell survival orproliferation, inhibitors of Mch6 can be used to treat diseasescharacterized by increased programmed cell death. Mch6 inhibitorsinclude, for example, small molecules and organic compounds that bindand inactivate Mch6 protease activity by a competitive ornoncompetitive-type mechanism, inhibitors of the conversion of inactiveproMch6 into active Mch6 protease or other molecules that indirectlyinhibit the Mch6 pathway. Such Mch6 inhibitors can include, for example,suicide inhibitors, anti-Mch6 antibodies and proteins, small peptidylprotease inhibitors, or small non-peptide organic molecule inhibitors.Specific examples of such inhibitors are described in Example II, andinclude substrate analogs such as tetrapeptide DEVD-CHO(Asp-Glu-Val-Asp-aldehyde; SEQ ID NO:81), DEVD-AMC(Asp-Glu-Val-Asp-aminomethylcoumarin; SEQ ID NO:82), YVAD-AMC(Tyr-Val-Ala-Asp-aminomethylcoumarin; SEQ ID NO:83), ZEVD-AMC(carbobenzoxy-Glu-Val-Asp-aminomethylcoumarin) and the cowpox virusprotein Crm A. Mch6 inhibitors can be formulated in a medium that allowsintroduction into the desired cell type or can be attached to targetingligands for introduction by cell-mediated endocytosis and otherreceptor-mediated events.

[0043] Mch6 inhibitors can also be used to treat or reduce the severityof diseases characterized by increased programmed cell death. In thisregard, Mch6 large subunits that lack the active site QACGG (SEQ IDNO:78) can be used to bind the small subunit of Mch6 to prevent theformation of active protease complexes. Such a mechanism of dominantnegative inhibition of Mch6 is similar to the dominant negativeinhibition of Ich-1_(L) by Ich-1_(S). Subunits from other ASCPs cansimilarly be used as dominant/negative inhibitors of Mch6 activity andtherefore be used to treat diseases mediated by programmed cell death.Such subunits should be selected so they bind either the p35 or p10 Mch6polypeptide and prevent their assembly into active tetrameric proteasecomplexes. Moreover, Mch6 subunits that have been modified to becatalytically inactive can also be used as dominant negative inhibitorsof Mch6. Such modifications include, for example, mutation of the activesite cysteine residue to include alanine or glycine, for example.

[0044] Mch6 substrate antagonists can similarly be used to treat orreduce the severity of diseases mediated by increased programmed celldeath. Such substrate antagonists can bind to and inhibit cleavage byMch6. Inhibition of substrate cleavage prevents commitment progressionof programmed cell death. Substrate antagonists include, for example,ligands and small molecule compounds.

[0045] Mch6 inhibitors can be identified by incubating cells with acompound to be tested for inhibitory activity and then treated with astimulus of apoptosis such as anti-Fas antibody, Fas ligand orstaurosporin. Control samples in the absence of inhibitors that aresubsequently treated with anti-Fas antibody undergo apoptosis andexhibit a rapid induction of ASCP activity. In contrast, samples in thepresence of inhibitors reduce or negate ASCP activity compared to thatin control samples.

[0046] Mch6 inhibitors can also be identified using Mch6-encodingnucleic acids and the Mch6 polypeptide of the invention in, for example,binding assays such as ELISA or RIA, or enzymatic assays usingtetrapeptide substrates, such as DEVD-AMC (SEQ ID NO:82) and YVAD-AMC(SEQ ID NO:83). DEVD-AMC (SEQ ID NO:82) and YVAD-AMC (SEQ ID NO:83)represent cleavage sites for the poly(ADP-ribose)polymerase (PARP) andIL-1β P1-P4 substrate tetrapeptides, respectively (Nicholson et al.,Nature 376:37-43 (1995)).

[0047] The Mch6 polypeptide to be used in such assays can be obtainedby, for example, in vitro translation, recombinant expression orbiochemical procedures. Such and other methods are known within the art,and are illustrated in Example II. For example, recombinant Mch6 can beexpressed by cloning Mch6 cDNA into a bacterial expression vector suchas pET21b (Novagen Inc., Madison, Wis.). The Mch6 can then be expressedand purified using routine molecular biology methods known to thoseskilled in the art. A purified recombinant Mch6 protein can be used tomeasure hydrolysis rates for various substrates, such as DEVD-AMC (SEQID NO:82) and YVAD-AMC in a continuous fluorometric assay.

[0048] Using such an assay for Mch6 activity, various compounds can bescreened for compounds that inhibit or enhance the expression of Mch6protease activity. Such screening methods are known to those skilled inthe art and can be performed by either in vitro or in vivo procedures.Such inhibitory molecules can be those contained in synthetic ornaturally occurring compound libraries. A specific example is phagedisplay peptide libraries where greater than 10⁸ peptide sequences canbe screened in a single round of panning.

[0049] Treatment or reduction of the severity of cell death-mediateddiseases can also be accomplished by introducing expressible nucleicacids encoding a Mch6 polypeptide or functional fragments thereof intocells characterized by such diseases. For example, elevated synthesisrates of Mch6 can be achieved by, for example, using recombinantexpression vectors and gene transfer technology. Similarly, treatment orreduction of the severity of cell death-mediated diseases can also beaccomplished by introducing and expressing antisense Mch6 nucleic acidsto inhibit the Mch6 synthesis rate. Such methods are well known withinthe art and are described below with reference to recombinant viralvectors. Other vectors compatible with the appropriate targeted cell canaccomplish the same goal and therefore can be substituted in the methodsdescribed herein in place of recombinant viral vectors.

[0050] Recombinant viral vectors are useful for in vivo expression of adesired nucleic acid because they offer advantages such as lateralinfection and targeting specificity. Lateral infection is inherent inthe life cycle of retroviruses and is the process by which a singleinfected cell produces many progeny virions that bud off and infectneighboring cells. The result is the rapid infection of a large area,most of which was not initially infected by the original viralparticles. This is in contrast to vertical-type infection in which theinfectious agent spreads only through daughter progeny. Viral vectorscan also be produced that are unable to spread laterally. Thischaracteristic can be useful if the desired purpose is to introduce aspecified gene into only a localized number of targeted cells.

[0051] Typically, viruses infect and propagate in specific cell types.Therefore, the targeting specificity of viral vectors utilizes thisnatural specificity to specifically introduce a desired gene intopredetermined cell types. The vector to be used in the methods of theinvention will depend on desired cell type to be targeted. For example,to treat neurodegenerative diseases by decreasing the Mch6 activity ofaffected neuronal cells, a vector should be used that is specific forcells of the neuronal cell lineage. Likewise, to treat diseases orpathological conditions of hematopoietic cells, a viral vector should beused that is specific for blood cells and their precursors, preferablyfor the specific type of hematopoietic cell. Moreover, such vectors canbe modified with specific receptors or ligands and the like to modify oralter target specificity through receptor-mediated events. Thesemodification procedures can be performed by, for example, recombinantDNA techniques or synthetic chemistry procedures. The specific type ofvector will depend upon the intended application. The actual vectors arealso known and readily available within the art or can be constructed byone skilled in the art using well known methodology.

[0052] Viral vectors encoding Mch6 nucleic acids or inhibitors of Mch6such as antisense nucleic acids can be administered in several ways toobtain expression of such sequences and therefore either increase ordecrease the activity of Mch6 in the cells affected by the disease orpathological condition. If viral vectors are used, for example, theprocedure can take advantage of their target specificity andconsequently need not be administered locally at the diseased site.However, local administration can provide a quicker and more effectivetreatment. Administration can also be performed by, for example,intravenous or subcutaneous injection into the subject. Injection of theviral vectors into the spinal fluid can also be used as a mode ofadministration, especially in the case of neurodegenerative diseases.Following injection, the viral vectors will circulate until theyrecognize host cells with the appropriate target specificity forinfection.

[0053] As described above, one mode of administration of Mch6-encodingvectors can be by direct inoculation locally at the site of the diseaseor pathological condition. Local administration is advantageous becausethere is no dilution effect and therefore a smaller dose is can besufficient to achieve Mch6 expression in a majority of the targetedcells. Additionally, local inoculation can alleviate the targetingrequirement associated with other forms of administration because avector can be used that infects all cells in the inoculated area. Ifexpression is desired in only a specific subset of cells within theinoculated area, then promoter and expression elements that are specificfor the desired subset can be used to accomplish this goal. Suchnon-targeting vectors can be, for example, viral vectors, viral genomes,plasmids, phagemids and the like. Transfection vehicles such asliposomes can be used to introduce the non-viral vectors described aboveinto recipient cells within the inoculated area. Such transfectionvehicles are known by one skilled within the art. Alternatively,however, non-targeting vectors can be administered directly into atissue of any individual. Such methods are known within the art and aredescribed by, for example, Wolff et al. (Science 247:1465-1468 (1990)).

[0054] Additional features can be added to the vectors to ensure safetyand/or enhance therapeutic efficacy. Such features include, for example,markers that can be used to negatively select against cells infectedwith the recombinant virus. An example of such a negative selectionmarker is the TK gene described above, which confers sensitivity to theantibiotic gancyclovir. Infection can therefore be controlled bynegative selection because it provides inducible suicide through theaddition of an antibiotic. Such protection ensures that if, for example,mutations arise that produce mutant forms of Mch6, dysfunction ofapoptosis will not occur.

[0055] It is understood that modifications that do not substantiallyaffect the activity of the various embodiments of this invention arealso included within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

EXAMPLE 1 Cloning And Characterization of Mch6

[0056] This Example shows the cloning, sequence analysis and tissuedistribution of Mch6. The results described herein indicate that Mch6 isa novel member of the ICE family of aspartate-specific cysteineproteases (ASCPs).

[0057] To identify potentially novel members of the ICE family of ASCPs,an approach combining information from the GenBank database of humanexpressed sequence tags (ESTs) and PCR was employed (seeFernandes-Alnemri et al., Cancer Res. 55:2737-2742 (1995);Fernandes-Alnemri et al., Cancer Res. 55:6045-6052 (1995)). Initially, asearch of GenBank ESTs for sequences related to CPP32 and Mch2identified a short EST sequence, which was used to derive a PCR primerto clone related cDNAs. The EST sequence was identified as accessionnumber T97582 and was used to design primer Mch6-pr1, having thenucleotide sequence CTCAACGTACCAGGAGCC (SEQ ID NO:3). A 10 μl aliquot ofhuman Jurkat λ Uni-Zap™ XR cDNA library (Fernandes-Alnemri et al., J.Biol. Chem. 269:30761-30764 (1994)) containing approximately 10⁸ pfu wasdenatured at 99° C. for 5 min. and used as a template for PCRamplification with the above Mch6-pr1 primer and a T3 vector-specificprimer (Stratagene).

[0058] A 10 μl aliquot of the primary amplification product was thenused as a template for a secondary PCR amplification with primerMch6-pr2, having nucleotide sequence CCTGGGAAAGTAGAGTAGG (SEQ ID NO:4),which was also derived from EST sequence T97582, and the SK-Zapvector-specific primer located downstream of the T3 primer, havingnucleotide sequence CAGGAATTCGGCACGAG (SEQ ID NO:5). The secondaryamplification products were cloned into a SmaI-cut pBluescript II KS⁺vector. The partial cDNA was then excised from the vector, radiolabeledand used to screen the original Jurkat λ Uni-Zap™ XR cDNA library forfull length cDNA clones. Positive clones were purified, rescued into thepBluescript II SK⁻ plasmid vector and sequenced.

[0059] The screen of the full length Jurkat λ Uni-Zap™ XR cDNA libraryresulted in the isolation of an approximately 2 kb cDNA clone. This cDNAcontains an open reading frame of 1248 bp (SEQ ID NO:1) that encodes a416-amino acid protein, named proMch6 (SEQ ID NO:2). As shown in FIG. 1,proMch6 is a polypeptide of 479 amino acid residues with a predictedmolecular mass of approximately 46.2 kDa.

[0060] Shown in FIG. 2 is a multiple amino acid sequence alignment ofrelatively conserved regions within the ASCPs. These regions include,for example, the relatively conserved active site pentapeptide sequenceQACRG (SEQ ID NO:79), residues involved in catalysis, residues that bindthe substrate-carboxylate of P1 Asp, residues adjacent to the substrateP2-P4 amino acids and the known and potential processing sites betweenthe small and large subunits. Sequence alignment of all known ASCPsrevealed that Mch6 belongs to the Ced-3-like subfamily of ASCPs.Briefly, previously identified ASCPs can be divided phylogeneticallyinto three subfamilies. The Ced-3-like ASCP subfamily includes Ced-3,CPP32, Mch2, Mch3, Mch4 and Mch5 (noncontiguous SEQ ID NOs:6-11). TheICE-like ASCP subfamily includes ICE, TX (noncontiguous ICH-2,ICErel-II, Mih1) and ICErelIII (noncontiguous SEQ ID NOs:12-14). TheNEDD-like subfamily includes ICH-1 (noncontiguous SEQ ID NO:15) and itsmouse counterpart NEDD2. Within the Ced-3-like subfamily, Mch6 shows thehighest homology to CPP32 (approximately 37% identity, 57% similarity).

[0061] Mch6 is also structurally similar to other ASCPs. The mature Mch6could be derived from the precursor proenzyme by cleavage at highlyconserved Asp residues (Asp315 and Asp330) located between the twosubunits. One difference between this enzyme and other family members,however, is that the fourth residue in its active site pentapeptidesequence QACGG (SEQ ID NO:78) is a Gly instead of Arg or Gln (FIG. 1).

[0062] Study of the known crystal structure of ICE has revealed thatHis237, Gly238 and Cys285 are involved in catalysis, while Arg179,Gln283, Arg341 and Ser347 are involved in binding the carboxylate sidechain of the substrate P1 aspartate (Walker et al., Cell 78:343-352(1994); Wilson, et al., Nature 370:270-275 (1994)). All these residuesare identical in all family members except in Mch5, where there is a Serto Thr conservative substitution for the residue corresponding to Ser347of ICE (FIG. 2). Another Ser to Thr conservative substitution can alsobe seen in Mch4 in the region corresponding to Ser236 of ICE, which isone of the residues that participates in binding the substrate P2-P4residues. Other residues that might participate in binding the substrateP2-P4 residues are not widely conserved, suggesting that they maydetermine substrate specificity.

[0063] In addition to the above sequence analysis, furthercharacterization of Mch6 was also performed by analyzing its tissuedistribution. This analysis was performed by RNA blot analysis of polyA⁺ RNA isolated from different human tissues (Fernandes-Alnemri et al.,Cancer Res. 55:6045-6052 (1995)). Briefly, tissue distribution analysisof Mch6 mRNA was performed on northern blots prepared by Clontech (SanDiego, Calif.) containing 2 μg/lane of poly A⁺ RNA from each tissue oforigin. A radioactive Mch6 riboprobe was prepared using Mch6 cDNA as atemplate for T7 RNA polymerase in the presence of [α³²P]ATP. The blotswere hybridized, washed and then visualized by autoradiography.

[0064] The proMch6 riboprobe detected three major mRNA species(approximately 1.0 kb, approximately 2.4 kb and approximately 4.4 kb) inmost human tissues. Highest expression was seen in the heart, andmoderate expression in the liver, skeletal muscle and pancreas. Lowestexpression was observed in the other tissues tested. The presence ofmultiple mRNA species has been observed with ICE and is suggestive ofalternative splicing or polyadenylation (Cerretti et al., Science256:97-100 (1992)).

[0065] To determine if Mch6 exhibits apoptotic activity, Sf9 baculoviruscells are used. Briefly, Sf9 cells are infected with recombinantbaculoviruses encoding full length Mch6, full length CPP32 or truncatedvariants of Mch6 or CPP32, separately or in various combinations. Cellsare then examined microscopically for morphological signs of apoptosissuch as blebbing of the cytoplasmic membrane, condensation of nuclearchromatin or release of small apoptotic bodies. In addition, the genomicDNA is examined for internucleosomal DNA cleavage.

EXAMPLE II Kinetic Parameters of Mch6

[0066] This Example characterizes the protease activity and substratespecificity of the ASCP Mch6.

[0067] The kinetic properties of bacterially expressed recombinant Mch6were determined using the tetrapeptide substrates DEVD-AMC (SEQ IDNO:82), ZEVD-AMC and YVAD-AMC (SEQ ID NO:83) in a continuousfluorometric assay (Table I). The DEVD-AMC (SEQ ID NO:82) and ZEVD-AMCrepresent the cleavage site for poly(ADP-ribose)polymerase (PARP). TheYVAD-AMC (SEQ ID NO:83) represents the cleavage site for IL-1β P1-P4substrate tetrapeptide (Nicholson et al., Nature 376:37-43 (1995)).Briefly, Mch6 cDNA was cloned in-frame into bacterial expression vectorpET21b (Novagen Inc., Madison, Wis.). The expression vector wasconstructed and expressed as a 6His-C terminal tagged protein in hostbacterial strain BL21(DE3) using routine molecular biology methods knownto those skilled in the art. After induction with IPTG, bacterialextracts were prepared from E. coli expressing the recombinant proteins.The extracts were then purified on a Ni⁺ affinity column.

[0068] The purified Mch6 protein was then used for further enzymaticanalyses. The activity of Mch6 was measured using bacterial lysatesprepared with ICE buffer (25 mM HEPES, 1 mM EDTA, 5 mM DTT, 0.1% CHAPS,10% sucrose, pH 7.5) at room temperature (24-25° C.). The K_(i) ofDEVD-CHO (SEQ ID NO:81) was determined from the hydrolysis rate of 50 AMDEVD-AMC (SEQ ID NO:82) following a 30 min preincubation of the enzymewith DEVD-CHO (SEQ ID NO:81). TABLE I Kinetic Parameters of Mch6Parameter Value K_(m)(DEVD-AMC)   10 μM K_(i)(DEVD-CHO) <10 nMV_(max)/K_(m) (DEVD-AMC) 100 V_(max)/K_(m) (ZEVD-AMC) 2.7 V_(max)/K_(m)(YVAD-AMC) <0.1

[0069] As shown above in Table I, Mch6 is potently inhibited by theDEVD-CHO peptide (K_(i)<10 nM). Mch6 also shows a greater than 1000-foldpreference for the CPP32-like substrate DEVD-AMC (SEQ ID NO:82) comparedto the ICE-like substrate YVAD-AMC (SEQ ID NO:83), by comparingV_(max)/K_(m) for each substrate. These kinetic parameters indicatesthat Mch6 is more related to the CPP32-like ASCPs than to the ICE-likeenzymes.

EXAMPLE III CPP32 Processes proMch6 in vitro

[0070] This example shows that CPP32 processes proMch6 at the Asp330site to yield a large and a small cleavage product.

[0071] Wild type proMch6 contains a potential CPP32 cleavage site at the³²⁷DQLD-A³³¹ site (SEQ ID NO:85), which is similar to the DEVD-G site(SEQ ID NO:86) in PARP and the DVVD-N site (SEQ ID NO:87) in proMch2(Nicholson et al., Nature 376:37-43(1995)). These similarities suggestthat CPP32 can also process proMch6 as well.

[0072] To provide proMch6 as a potential processing substrate, proMch6cDNA was transcribed and translated in vitro in the presence of³⁵S-methionine using coupled transcription/translation TNT kit accordingto the manufacturer's recommendations (Promega, Madison Wis.). Twomicroliters of the translation reaction were incubated with purifiedenzymes (100-200 ng) or bacterial lysates in ICE buffer (25 mM HEPES, 1mM EDTA, 5 mM DTT, 0.1% CHAPS, 10% sucrose, pH 7.5), in a final volumeof 10 μl. The reaction was incubated at 37° C. for 1-2 hours and theresulting translation products were analyzed by Tricine-SDS-PAGE andautoradiography.

[0073] To determine the ability of CPP32 to process proMch6, proMch6from in vitro translation was incubated for various times with purifiedrecombinant human CPP32 and then analyzed by Tricine-SDS-PAGE andautoradiography. The CPP32 was obtained by expression in bacteria,assayed for activity and purified on a Ni⁺² affinity resin(Fernandes-Alnemri et al., Cancer Res. 55:2737-2742; Fernandes-Alnemriet al., Cancer Res. 55:6045-6052 (1995)).

[0074] Analysis of the time course revealed that CPP32 cleaves proMch6at one site to generate two cleavage products of approximately 37 kDa orp37 and approximately 10 kDa or p10. The sizes of the products wereconsistent with cleavage at the ³²⁷DQLD-A³³¹ site (SEQ ID NO:85), whichcontains Asp330. Products were initially detected within the first 15minutes. Longer incubation resulted in decreased intensity of the fulllength 46 kDa band and increased intensity of the p37 and p10 products.Prolonged incubation did not result in significant processing of the p37product. This result indicates that CPP32 cleaves preferentially at onlyone site within proMch6.

[0075] The CPP32 processing site was confirmed by constructing andanalyzing mutants of proMch6. Briefly, potential processing sites weremutated by site-directed mutagenesis using overlapping PCR mutagenicoligonucleotides. The resulting PCR products were subcloned inpBluescript II KS⁺ vector under the T7 promoter and their sequences wereverified by DNA sequencing. In particular, Asp315 of proMch6 was mutatedto Ala for one mutant and Asp330 was mutated to Ala for another mutant.

[0076] As with the wild type proMch6, the Asp315 mutant was processed byCPP32 to generate the p37 and p10 products. In contrast, CPP32 failed toprocess the Asp330 mutant. These results show that CPP32 processesproMch6 at the Asp330 site.

EXAMPLE IV Granzyme B Cleaves proMch6 Preferentially at the PEPD-A site(SEQ ID NO:84)

[0077] This example shows that granzyme B cleaves proMch6 at two sites,with a preference for cleaving at the ³¹²PEPD-A³¹⁶ site (SEQ ID NO:84).

[0078] Granzyme B can induce apoptosis in target cells by activation ofASCPs. The ability of granzyme B to process several members of the ASCPfamily has now been demonstrated (Fernandes-Alnemri et al., Proc. Natl.Acad. Sci. USA 93:7464-7469 (1996)). To test whether granzyme B canprocess proMch6, ³⁵S-labeled proMch6 was prepared and incubated withgranzyme B and then analyzed by Tricine-SDS-PAGE and autoradiography, asdescribed in Example III. The granzyme B used for these assays waspurified by immunopurification from human natural killer cell lysatesusing granzyme B-specific monoclonal antibody (Trapani et al., Biochem.Biophys. Res. Commun. 195:910-920 (1993); Trapani et al., J. Biol. Chem.269:18359-18365 (1994)). The results indicate that granzyme B cleavesproMch6 preferentially at one site to generate a large product ofapproximately 35 kDa (p35) and a small product of approximately 12 kDa(p12).

[0079] In comparison with the products of CPP32 processing described inExample III, the large granzyme B product (p35) migrates faster than thelarge CPP32 product (p37), indicating that the enzymes cleave at twodifferent sites. The granzyme B cleavage site is in the N-terminaldirection of the CPP32 cleavage site and it is most likely to be Asp315within the ³¹²PEPD-A³¹⁶ site (SEQ ID NO:84).

[0080] The location of the granzyme B cleavage sites were confirmed byusing proMch6 mutants as described in Example III. Unlike CPP32,granzyme B cleaved the Asp330 mutant to generate p35 and p12 products.When granzyme B was assayed with the Asp315 mutant, the p35 and p12products were absent. This result indicated that granzyme B was unableto process the ³¹²PEPD-A³¹⁶ site (SEQ ID NO:84). Granzyme B did cleavethe Asp315 mutant at the ³²⁷DQLD-A³³¹ site (SEQ ID NO:85) to generatefaint p37 and p10 products, albeit inefficiently. A double mutant formof proMch6 at Asp315 and Asp330, which was prepared by site-directedmutagenesis as described in Example III, completely blocked processingof proMcH6 by granzyme B and CPP32. These results show that granzyme Bprocesses proMch6 at Asp315 and Asp330 with preference for Asp315 overAsp330.

EXAMPLE V Activation of proCPP32 by Granzyme B in a Cell Lysate Lead toProcessing of proMch6 at Asp315 and Asp330 Simultaneously

[0081] This example shows that activation of proCPP32 by granzyme B incell lysate results in cleavage of proMch6 at two sites simultaneously.

[0082] As described in Examples III and IV, CPP32 and granzyme B processproMch6 at two different sites. To determine whether granzyme B canprocess proCPP32 into CPP32, followed by simultaneous processing ofproMch6 by granzyme B and CCP32, ³⁵S-labeled proMch6 was mixed with cellextract from 697 lymphocyte cell line and then incubated with granzymeB.

[0083] The products of the incubated reaction were analyzed at differenttime points by western blotting to detect activation of endogenousproCPP32. The western blots used a rabbit polyclonal anti-human CPP32antibody, which was raised against recombinant p20 subunit (amino acids1-175) of human CPP32. Autoradiography was also used to detectprocessing of proMch6. The p20/p19 doublet of mature CPP32 was detectedin less than 15 minutes, simultaneously with the disappearance of the 32kDa proCPP32. Subsequently, the p20 product was autocatalyticallyprocessed to the p19. In the presence of the DEVD-CHO (SEQ ID NO:81)inhibitor, only the p20 could be detected due to the inhibition of CPP32under these conditions.

[0084] Autoradiographic analysis of the same samples revealed atime-dependent processing of proMch6 to the p35 (granzyme B product) andp37 (CPP32 product) bands. Also detectable was the p12/p10 band. WhenCPP32 was inhibited by the DEVD-CHO (SEQ ID NO:81), only the granzymeB-generated p35 and p12 bands were detected.

[0085] The above results indicate that the p37, p35, p12 and p10products are produced in cells undergoing granzyme B-mediated apoptosis.This interpretation is based on the fact that cell lysates contain allthe cytoplasmic components necessary for apoptosis (Trapani et al.,Biochem. Biophys. Res. Commun. 195:910-920 (1993); Martin, et al., EMBOJ. 14:5191-5200 (1995); Enari et al., EMBO J. 14:5201-5208 (1995)).Therefore, the mature Mch6 enzyme is derived from the proenzyme bycleavage at Asp315 and Asp330 to generate the large (p35) and small(p10) subunits, as shown in FIG. 3. Due to the lack of evidence ofefficient processing in the prodomain in cell lysates, these resultsindicate that the p35 is the large subunit and p10 is the small subunitof mature Mch6. Moreover, these results further show that activation ofproCPP32 by granzyme B in cell lysate results in cleavage of proMch6 attwo sites simultaneously.

[0086] Throughout this application, various publications are referencedwithin parentheses. The disclosures of these publications in theirentireties are hereby incorporated by reference in this application inorder to more fully describe the state of the art to which thisinvention pertains.

[0087] Although the invention has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific experiments detailed are only illustrative of theinvention. It should be understood that various modifications can bemade without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims.

What is claimed is:
 1. An isolated gene encoding Mch6, or functionalfragment thereof.
 2. The isolated gene of claim 1 , comprisingsubstantially the coding sequence in SEQ ID NO:1.
 3. The isolated geneof claim 1 , wherein the functional fragment comprises single or doublestranded nucleic acids of the sequence shown in SEQ ID NO:1.
 4. Theisolated gene of claim 1 , wherein the functional fragment comprisescoding or non-coding strands of the sequence shown in SEQ ID NO:1.
 5. Anisolated nucleic acid sequence encoding Mch6, comprising substantiallythe sequence shown in SEQ ID NO:1, or functional fragment thereof. 6.The isolated nucleic acid sequence of claim 5 , wherein the functionalfragment comprises single or double stranded nucleic acids of thesequence shown in SEQ ID NO:1.
 7. The isolated nucleic acid sequence ofclaim 5 , wherein the functional fragment comprises coding or non-codingstrands of the sequence shown in SEQ ID NO:1.
 8. An isolated Mch6polypeptide, comprising substantially the amino acid sequence shown inSEQ ID NO:2, or functional fragment thereof.
 9. The isolated Mch6polypeptide of claim 8 , wherein the functional fragment furthercomprises a catalytic domain of the protease.
 10. An isolated largesubunit of Mch6 polypeptide shown in SEQ ID NO:2, or functional fragmentthereof.
 11. An isolated small subunit of Mch6 polypeptide shown in SEQID NO:2, or functional fragment thereof.